Microscope for high spatial resolution imaging a structure of interest in a sample

Abstract

A microscope for high spatial resolution imaging a structure of interest in a sample comprising a substance having a first state with first spectral properties and a second state with second spectral properties, the microscope comprising: an objective-lens assembly, a wave front modulating optical device adapted to spatially vary an intensity of a transfer light beam, a probe detector arranged to detect an optical measurement signal from a portion of the substance in the second state and placed in an area of the transfer light beam with an intensity adapted not to transfer the substance between the first and second states said microscope comprising a phase contrast microscopy system which includes an intensity detector arranged to detect an intensity of an illuminating light beam after said illuminating light beam has passed through the sample, the objective-lens assembly and the wave front modulating optical device.

The invention claimed is: 
     
       1. A microscope for high spatial resolution imaging a structure of interest in a sample comprising a substance, said substance having a first state with first spectral properties and a second state with second spectral properties, the microscope comprising:
 an objective-lens assembly having first and second sides opposite to each other, and presenting first and second focal planes respectively on said first and second sides, the sample being intended to be placed on the first side, 
 a transfer light source arranged on the second side of the objective-lens assembly to emit a transfer light beam towards the sample through the objective-lens assembly, said transfer light beam having an intensity and a phase profile, 
 a light beam modulating device placed between the transfer light source and the objective-lens assembly, said light beam modulating device being adapted to spatially vary the transfer light beam so that said transfer light beam after having passed trough said light beam modulating device and the objective-lens assembly presents at least a first intensity area with a first intensity adapted to transfer the substance in the second state to the first state, and at least a second intensity area with a second intensity adapted not to transfer the substance between the first and second states, 
 a probe detector adapted to detect an optical measurement signal from a portion of the substance in the second state, 
 wherein said microscope comprises a phase contrast microscopy system which includes: 
 the light beam modulating device further adapted to generate a phase contrast, 
 an illuminating light source arranged to emit an illuminating light beam towards the sample, said illuminating light beam having an intensity and a phase profile, 
 an intensity detector arranged to detect the intensity of the illuminating light beam after said illuminating light beam has passed at least once through the sample, the objective lens assembly and the light beam modulating device. 
 
     
     
       2. The microscope according to  claim 1 , wherein the light beam modulating device is adapted to vary the transfer light beam so that the first intensity area surrounds the second intensity area. 
     
     
       3. The microscope according to  claim 1 , wherein the light beam modulating device being a phase mask adapted to modulate the phase profiles of the transfer light beam and of the illuminating light beam. 
     
     
       4. The microscope according to  claim 3 , wherein the light beam modulating device is a spiral phase mask adapted to apply an angular-dependent phase shift to the phase profiles of the transfer light beam and of the illuminating light beam. 
     
     
       5. The microscope according to  claim 1 , wherein the substance comprises a fluorescent marker emitting fluorescence when relaxing from the second state to the first state, the microscope further comprising a probing light source adapted to emit a probing light beam overlaid with the transfer light beam, said probing light beam having an intensity adapted to transfer the marker in the first state to the second state, the transfer light beam in the first intensity area being adapted to deplete the second state of the marker. 
     
     
       6. The microscope according to  claim 5 , wherein the transfer light source and the probing light source form a single light source which produces both transfer and probing light beams. 
     
     
       7. The microscope according to  claim 1 , further comprising a transfer light scanning arrangement adapted to move the transfer light beam and the sample relative to each other so that the sample can be scanned by the transfer light beam. 
     
     
       8. The microscope according to  claim 1 , further comprising an illuminating light scanning arrangement adapted to move the illuminating light beam and the sample relative to each other so that the sample can be scanned by the illuminating light beam. 
     
     
       9. The microscope according to  claim 1 , wherein the intensity detector is a pixelated detector. 
     
     
       10. The microscope according to  claim 1 , wherein the intensity detector is a single pixel detector. 
     
     
       11. The microscope according to  claim 1 , wherein the illuminating light source comprises a coherent light source. 
     
     
       12. The microscope according to  claim 1 , wherein the illuminating light source comprises an incoherent light source. 
     
     
       13. The microscope according to  claim 1 , wherein the illuminating light source is arranged on the first side of the objective-lens assembly at a distance defining a space for placing the sample. 
     
     
       14. The microscope according to  claim 13 , wherein the intensity detector is arranged on the second side of the objective-lens assembly. 
     
     
       15. The microscope according to  claim 1 , wherein the illuminating light source is arranged on the second side of the objective-lens assembly. 
     
     
       16. The microscope according to  claim 15 , wherein a reflective element is arranged on the first side of the objective-lens assembly at a distance defining a space for placing the sample. 
     
     
       17. The microscope according to  claim 15 , wherein the intensity detector is arranged on the first side of the objective-lens assembly. 
     
     
       18. The microscope according to  claim 15 , wherein the transfer light beam is the illuminating light beam. 
     
     
       19. The microscope according to  claim 9 , wherein the pixelated detector is a CCD camera. 
     
     
       20. The microscope according to  claim 9 , wherein the pixelated detector is a CMOS camera. 
     
     
       21. The microscope according to  claim 10 , wherein the single pixel detector is an avalanche photo-diode. 
     
     
       22. The microscope according to  claim 10 , wherein the single pixel detector is a photomultiplier. 
     
     
       23. The microscope according to  claim 11 , wherein the coherent light source is a laser device. 
     
     
       24. The microscope according to  claim 12 , wherein the incoherent light source is a light-emitting diode. 
     
     
       25. The microscope according to  claim 12 , wherein the incoherent light source is a mercury vapour lamp. 
     
     
       26. The microscope according to  claim 12 , wherein the incoherent light source is a halogen lamp.